The UNC-45 protein chaperone is a member of the UNC-45/CRO1/She4p (UCS) family. Its loss results in abnormal skeletal and cardiac muscle development in several animal models, suggesting a conserved evolutionary function. UNC-45 interacts with chaperones Hsp90 and Hsp70 through its N-terminal TPR domain and with the myosin motor region through its C-terminal UCS domain. While its mechanism is unclear, UNC-45 appears to act as both scaffold and chaperone during myosin motor folding. Alterations in UNC-45 expression levels are associated with inclusion body myopathy and cardiac failure, linking UNC-45 to human disease. Recently, the first human UNC-45 mutant was shown to cause juvenile cataracts. Finally, our group recently found that UNC-45 is a viable therapeutic in protein folding diseases. The goal of this proposal is to expand our understanding of UNC-45 function and its mechanism of action by identifying novel UNC-45 interacting partners using complementary genetic and biochemical approaches. To this end, we crossed chromosomal-segment haploid deficiency lines onto an UNC-45 haploid (sensitized) background and screened flight ability as a readout of muscle function. We identified several candidate deficiency lines that exhibit poor flight ability in conjunction with haploid loss of UNC-45. Aim 1 of this project will utilize the powerful mutant allele and RNAi knockdown techniques in Drosophila to map specific genes that exhibit defective muscle development in the UNC-45 sensitized background. This will test the hypothesis that candidate chromosomal-segment deficiency lines harbor specific genes whose products functionally interact with UNC-45 during muscle development. Aim 2 will employ a complementary biochemical approach to isolate and identify UNC-45 interacting partners using co-immunoprecipitation followed by mass spectrometry. A transgenic fly expressing an UNC-45:GFP fusion protein will allow us to capture and analyze UNC-45 complexes formed during fly development and organism stress. This will test the hypothesis that UNC-45 interacting partners change during muscle development and stress. Aim 3a will localize identified UNC-45 interacting partners in vivo using confocal microscopy and will use RNAi knockdown to assess their roles in muscle development. Aim 3b will functionally interrogate the ability of identified partners to modulate UNC-45 chaperone activity using myosin aggregation protection and citrate synthase folding assays. We will also investigate whether identified proteins act as UNC-45 client proteins using heat-induced aggregation protection assays. Aim 3 will address the hypothesis that UNC-45 interacting partners affect UNC-45 chaperone activity or act as UNC-45 clients within muscle cells. Overall, our project will provide an integrative analysis of UNC-45 interacting partners and their functional roles in UNC-45 biology during muscle development. It will offer outstanding training in Drosophila genetics, cell biology and biochemistry as well as mentoring in scientific writing, research lab supervision and teaching.